Molecular Study of Lycopene Effect on Clinical Isolates of Cryptococcus Neoformans

Cryptococcosis is the most common cause of invasive fungal infections, especially cryptococcal meningitis [1]. Since the 1970s, Cryptococci species of Gatti and neoformans are the commonest problematic Cryptococcosis in humans, especially [2]. Meningitis caused by Cryptococcus is reported to have a significant attributable mortality and morbidity among immunocompromised patients, especially in association with AIDS [3]. Cryptococcus is a yeast belonging to basidiomycetes. This genus has more than 30 species are sharing in many environmental features; they differ in characteristics due to the geographic distributions, hosts and their immune state and clinical features [4]. The patients with cryptococcal meningitis experience headache, fever, cranial problems, memory loss and irritation. These symptoms may be more acutely or less as absence of headache [5,6]. Cutaneous cryptococcosis is rare infection and classified as third commonest expressing of cryptococcus infections, it occurs either associated with another skin disease or by direct inoculation of this yeast. The symptoms of cutaneous cryptococcosis interacts with many skin lesions. Therefore, it needed to culture of skin biopsy for adequate diagnosis [7]. The treatment of cryptococcosis based on deficiency of virulence factors, as well as using of anti-cryptococcal drugs and assessment their activity by molecular study of virulence genes of causative agents of cryptococcosis and find alternative treatments for resistance isolates [8].

Lycopene is a carotenoid compound, it occurring in nature in some red colored plants like tomatoes, pink grapefruit, watermelon, papaya and guava [9]. The human cell unable to synthesize lycopene. Thus, it supplemented through the daily diet and the age of humans is directly proportion with their needed for lycopene and the deficiency of this carotenoid association with some pathological problems such as cutaneous, cardiovascular and neurological diseases [10,11].

Samples Collections

One hundred samples as total were collected from patient (males and females) whom admitted different teaching hospitals in Baghdad province for a year period (2023). The distribution specimens were possessed from patient respiratory infections and burn (40 each) and 20 from patients with meningitis.

The fruits of Tomatoes (Lycopersicum esculentum) were possessed during summer of locally market at Baghdad, Iraq. Firstly, the fruits lotion using distill water and sterilized using ethanol (70%). Next, they dried using oven with two periods (at 90֯ C for 90 minutes and at 60֯ C for 240 minutes). Then, the fruits were cut to cubic pieces with dimensioned (10×10×10) mm³ and grinded in according to [12,13]. After that, the grinded fruits were packed in darkled test tubes and kept at room temperature until chemical extraction of lycopene.

Identification of Cryptococcus Neoformans

Cultural Identification: All the collecting specimens were streaked directly onto SDA medium with incubation at 37°C for seven days with daily examination. For microscopic examination, a loopful of C. neoformans colonies were placed onto sterilized slide and added a drop of Indian ink on the yeast with microscopic examination under (40x) to reveal capsules presence [6,14]. The isolated yeasts were macroscopic and microscopic identified based on their morphological characteristics [15].

Chemical extraction of Lycopene

The extraction of lycopene was performed according to [16]. Ten grams of dried tomatoes and put in darkled tubes to prevent light oxidation. Next, 100 ml from ethanol were added with shaking with using vortex for 30 minutes. Then, the tubes were left for 2 minutes and separate lycopene with filter solution under vacuum. After that, the extracted lycopene was place in a sterilize Petri dish and dried using anhydrous calcium chloride by vacuum desiccators.

Lycopene Purification

High performed liquid chromatography (HPLC) was used for Lycopene purification and as mentioned in [17]. The purification circumstances of HPLC were: columns were supplied they Chromoleth RP−C18A, 4.0×10 mm, injection volumes (40 μls) at 25°C, flows range (1cm³ ⁄min), waves length (470 nm), solvents which used dimethylforomide (50%) and acetonitril (30%).

Detection of Lycopene Effect as Antifungal

The antifungal effect of purified lycopene was done using well diffusion technique [18,19]. Firstly, the isolates of C. neoformans were streaked onto Muller Hinton Agar medium. Next, the wells (5 mm in diameter) were done onto using a sterilized cork borer. After that, one hundred microliters of purified lycopene with concentrations (10,20,40 and 80 mg/ml) were spread into the wells. Finally, the cultured Petri dishes were incubated at 37⁰C for 24-48 hours.

Molecular Identification

DNA of six isolates of C. neoformans were possessed by Geneaid DNA extraction kit, Thailand, based on instructions of manufacture. CAP60 gene detected by forward primers CAP60F: 5`-GCAGCGGCTTGCCATTCGTG-3` and reversed primers (CAP60R: 5`-AGTCCGTGGAGGCGTGGTCA-3`) [15]. PCR amplifications of CAP60 gene were done in 25μl containing 3 μL of C. neoformans DNA, 10μl Taq Pol PCR Pre master Mix ,2μls of each primer (20pmol) and 8 μls of nucleases free water were add onto tubes till achieve 25 μL. PCR was done by thermos-cycler (Amersha, Sweden) depending on PCR program revealed by Alarousy et al. [16]. In a brief, the amplification of CAP60 gene of C. neoformans was done with initial denaturations at 94ºC for 4 mins follow 30 cycles PCR. Denaturation at 94ºC for 60 secs, annealings at 50ºC for 45 secs, extensions at 72ºC for 2 minutes and thermal cycles were terminated by final extensions at 72 °C for 5 mins. Nucleases free water use as a control; a 1.5 % agarose gel was used to analyze PCR product of CAP60 gene after it stained with ethidium bromide and detected using UV transilluminator. To reveal the genetic alterations of single nucleotide base that replaced by another, gene sequencing method was done for detection of substitution mutations of CAP60 gene for treated and untreated C. neoformans with lycopene.

The isolates were sent to Microgen company (South Korea) for CAP60 gene sequencing in both direction and all the reference nucleotide sequences of this gene from (www.ncbi) and aligned using MEGA4 program.

Percentage of Morphologically Identified Cryptococcus Neoformans

The identification of C. neoformans isolates was based on the macroscope and microscopic examinations. Macroscopic examination of C. neoformans isolates that were cultured on SDA media incubated at 37°C for 48 hours appears as whitish to creamy in colour with spherical, mucoid and smooth yeast cells. Whereas, Microscopic examination appeared Globule to ovoid budded yeast cells with surrounding by a gelatinous capsule, as in Figure 1(a,b).

Figure 1(a,b): (a) Macroscopic features of C. neoformans colonies cultured on SDA medium and (b) Microscopic features of C. neoformans yeast stained with LPCB (40X)

The positive percentages of C. neoformans among patients was 9% (9 out of 100) as shown in Table 1.

Table 1: Percentage of C. neoformans isolates concerning types of infection

This result agreed with Inaam et al. [20] and Al-Temimay and Esraa [21]. Cryptococcus neoformans is an opportunistic yeast with higher causative agent of morbidity and mortalities in immunocompromised than immunocompetent patients [22].

Effect of Purified Lycopene on Cryptococcus Neoformans Isolates

Three out of nine isolates of C. neoformans (33.3%) were sensitive to purified lycopene (40 mg\ ml) with ascending inhibition zones (4,9 and 13) mm. Harishawi et al. [18] found that the inhibition zones (6,7,8 and 9) mm, respectively, for C. neoformans as shown in Figure 2.

Figure 2: Effect of purified lycopene on C. neoformans isolates

The active compounds of lycopene with structural and chemical structures had excessive antimicrobial and biological activities [20].

Molecular Detection of the CAP60 Gene of Cryptococcus Neoformans Isolates

The molecular weight of CAP60 gene was 600 bp., this was indicated sign for successes reaction, as in Figure 3.

Figure 3: Gel electrophoresis of CAP60 gene product for C. neoformans using 2% agaroses gel at 6V/cm for 60 minutes. M: 100 DNA ladder, lanes 1-3: uniplex PCR products

After performing of the alignment between the amino acid reference sequences of CAP60 gene of C. neoformans and amino acids sequences of three isolates treated with lycopene and three isolates untreated with lycopene, two isolates of that treated with lycopene were mutant which have more than one substitution mutations, like in Figure 4.

Figure 4: The substitution mutations of C. neoformans isolates that were treated with purified lycopene extract.

There are no published results to compare with it. C. neoformans isolates require the CAP60 gene for capsule formation, which plays vital roles in isolates' virulence [8].

This study concluded that the gene sequencing of the CAP60 gene revealed that the treated isolates with lycopene had more than substitution mutations.

Ethical Statement

The information form of this study was confirmed by local ethic committee based on the decision 1828 in 12\1\2024.

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